Chronic myeloid leukemia (CML) is characterized in the majority of cases by a t(9;22)(q34;q11) translocation, also called the Philadelphia chromosome, giving rise to the BCR-ABL1 fusion protein. Current treatment with tyrosine kinase inhibitors is directed against the constitutively active ABL1 domain of the fusion protein, and minimal residual disease (MRD) after therapy is monitored by real-time quantitative PCR (RQ-PCR) of the fusion transcript. Here, we describe a novel approach to detect and enumerate cells positive for the BCR-ABL1 fusion protein by combining the in situ proximity ligation assay with flow cytometry as readout (PLA-flow). By targeting of the BCR and ABL1 parts of the fusion protein with one antibody each, and creating strong fluorescent signals through rolling circle amplification, PLA-flow allowed sensitive detection of cells positive for the BCR-ABL1 fusion at frequencies as low as one in 10,000. Importantly, the flow cytometric results correlated strongly to those of RQ-PCR, both in diagnostic testing and for MRD measurements over time. In summary, we believe this flow cytometry-based method can serve as an attractive approach for routine measurement of cells harboring BCR-ABL1 fusions, also allowing simultaneously assessment of other cell surface markers as well as sensitive longitudinal follow-up.

Detection of cells expressing BCR-ABL1 with PLA-flow. The assay employs a pair of oligonucleotide-conjugated antibodies (PLA probes) with affinity for the BCR and ABL1 domains of the fusion protein in fixed and permeabilized cells (A). The oligonucleotides on PLA probes remaining in close proximity after washes serve as templates for hybridization of two additional DNA oligonucleotides, guiding their ligation into DNA circles (B). After ligation, the DNA circles are locally amplified by rolling circle amplification (RCA) to generate RCA products that are visualized and detected by addition of complementary fluorophore-labeled oligonucleotides (C), followed by detection of labeled cells via flow cytometry.